Project Summary Cellular inclusions of proteins are primary hallmarks of a great majority of neurodegenerative diseases. In common forms of amyotrophic lateral sclerosis (ALS), Alzheimer?s disease related dementias (frontotemporal dementia; limbic-predominant age-related TDP-43 encephalopathy (LATE)) as well as some forms of Alzheimer?s disease, the essential human TAR DNA binding protein of 43 kDa (TDP-43) forms intraneuronal aggregates. Importantly, dozens of missense mutations in an aggregation-prone domain of TDP-43 have been found in familial and sporadic cases of ALS and frontotemporal dementia. These data provide strong support for the direct causative pathological role for TDP-43 in neurodegeneration in Alzheimer?s disease related dementias and motor neuron disease. Additionally, recent research modulating the TDP-43 interactome demonstrates that TDP-43 is an important potential therapeutic targets in these diseases. However, therapeutic development is hampered in large part by an absence of mechanistic understanding of normal TDP-43 function, the molecular effect of the mutations causing amyotrophic lateral sclerosis and frontotemporal dementia, and the TDP-43 disruption in neurodegenerative disease. These gaps are due in large part to a lack of atomic structural data regarding TDP-43, its complexes, and its conversion to aggregates, which in turn is due to the difficulty in observing TDP-43 complexes via traditional structural biology techniques. This project will make use of integrated experimental and computational structural biology techniques combined with molecular and cell biology approaches to 1) determine the atomistic details of the assembly of a helical sub-region of TDP-43, its contribution to splicing function, and its structural conversion in disease aggregates, 2) identify how known and novel post-translational modifications and disease-associated mutations alter TDP-43 self- and hetero-protein contacts that mediate and regulate TDP-43 liquid-liquid phase separation and disease-associated aggregation, and 3) map the structural basis of the interactions of TDP-43 with poly(ADP-ribose) and importin machinery that serve as promising therapeutic targets. The challenging, dynamic, structural targets necessitate the approach highlighting a tight connection between molecular simulation and experimental biophysical techniques (primarily NMR spectroscopy). These approaches will generate detailed molecular models of the interactions that will be tested for functional relevance using in cell aggregation, in cell splicing, and in cell protein/RNA binding structure (iCLIP). The results of these studies on TDP-43 complexes, phase-separation, function, and aggregation will provide direct structural and mechanistic input to the design of strategies to prevent toxic disruption of TDP-43 in Alzheimer?s disease related dementias and motor neuron disease. These insights represent potential for future treatments for ALS, frontotemporal dementia, and other TDP-43-associated diseases that currently have no cure or effective treatments.